Targeted disruption of t cell receptor genes using engineered zinc finger protein nucleases

ABSTRACT

Disclosed herein are methods and compositions for inactivating TCR genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/173,875, filed Oct. 29, 2018, now U.S. Pat. No. 11,439,666,which is a continuation of U.S. patent application Ser. No. 14/591,625,filed Jan. 7, 2015, now U.S. Pat. No. 10,155,011, which is acontinuation of U.S. patent application Ser. No. 12/927,292, filed Nov.10, 2010, now U.S. Pat. No. 8,956,828, which claims the benefit of U.S.Provisional Application Nos. 61/280,863, filed Nov. 10, 2009;61/404,064, filed Sep. 27, 2010 and 61/404,685, filed Oct. 6, 2010, thedisclosures of which are hereby incorporated by reference in theirentireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on May 12, 2023, isnamed 128687-2851.xml and is 153,395 bytes in size.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the field of genome modification of humancells, including lymphocytes and stem cells.

BACKGROUND

Various methods and compositions for targeted cleavage of genomic DNAhave been described. Such targeted cleavage events can be used, forexample, to induce targeted mutagenesis, induce targeted deletions ofcellular DNA sequences, and facilitate targeted recombination at apredetermined chromosomal locus. See, for example, U.S. PatentPublication Nos. 2003/0232410; 2005/0208489; 2005/0026157; 2005/0064474;2006/0188987; 2008/015996, and International Patent Publication No. WO2007/014275, the disclosures of which are incorporated by reference intheir entireties for all purposes.

The T cell receptor (TCR) is an essential part of the selectiveactivation of T cells. Bearing some resemblance to an antibody, the TCRis typically made from two chains, α and β, which co-assemble to form aheterodimer. The antibody resemblance lies in the manner in which asingle gene encoding a TCR chain is put together. TCR chains arecomposed of two regions, a C-terminal constant region and an N-terminalvariable region. The genomic loci that encode the TCR chains resembleantibody encoding loci in that the TCR α gene comprises V and Jsegments, while the β chain locus comprises D segments in addition to Vand J segments. During T cell development, the various segmentsrecombine such that each T cell has a unique TCR structure, and the bodyhas a large repertoire of T cells which, due to their unique TCRstructures, are capable of interacting with unique antigens displayed byantigen presenting cells. Additionally, the TCR complex makes up part ofthe CD3 antigen complex on T cells.

During T cell activation, the TCR interacts with antigens displayed onthe major histocompatability complex (WIC) of an antigen presentingcell. Recognition of the antigen-WIC complex by the TCR leads to T cellstimulation, which in turn leads to differentiation of both T helpercells (CD4+) and cytotoxic T lymphocytes (CD8+) in memory and effectorlymphocytes. These cells then can expand in a clonal manner to give anactivated subpopulation within the whole T cell population capable ofreacting to one particular antigen.

Cytotoxic T lymphocytes (CTLs) are thought to be essential in killingtumor cells. These cells typically are able to induce apoptosis incancer cells when the cancer cell displays some antigen on its surfacethat was previously displayed on the WIC by an antigen presenting cell.Normally, following action against target cells, CTLs will apoptose whenthe cellular threat is cleared, with a subset of lymphocytes remainingthat will further differentiate into memory T cells to persist in casethe body is exposed to the antigen again. The pool of memory lymphocytesis possibly highly heterogeneous. Recently, two types of memory T-cellshave been identified: effector memory T-cells (CD45RA-CCR7-, CD62L-) andcentral memory T-cells that are CD45RA negative cells characterized bythe expression of CCR7 and CD62L, two molecules required for homing inT-cell areas of secondary lymphoid organs. Upon antigenic stimulation,central memory T-cells produce low levels of effector cytokines such asIL-4 and IFN-γ, but high levels of IL-2, which is able to sustain theirrapid and consistent proliferation. Upon antigen encounter centralmemory T-cells undergo: 1) proliferation, resulting in anauto-regenerative process, aimed at increasing their pool, and 2)differentiation, resulting in the generation of effector memory T-cells,which are characterized by a low proliferative potential but are able tomigrate to inflamed non-lymphoid tissues and mediate the effector phaseof the immune response. Protocols enabling gene transfer into Tlymphocytes, while preserving their central memory functional phenotypehave been developed (see European Patent Publication No EP1956080,Kaneko, et al. (2009) Blood 113(5):1006-15).

However, some tumor cells are able to escape surveillance by the immunesystem, perhaps through mechanisms such as poor clonal expansion ofcertain CTL subsets expressing the relevant TCR, and localized immunesuppression by cancer cells (see Boon, et al. (2006) Annu Rev Immunol.24:175-208). The notion of a cancer vaccine is built upon the idea ofusing these cancer specific antigens to stimulate and expand the CTLsthat express the appropriate TCR in vivo, in an attempt to overcomeimmune escape, however, these cancer vaccines have yet to show anymarked success. In fact, an analysis done in 2004 examined 765metastatic cancer patients that had been treated in over 35 differentcancer vaccine trials, where an overall response was observed in only3.8% of patients (see Rosenberg, et al. (2004) Nat. Med. 10(9):909-915).

Adoptive immunotherapy is the practice of achieving highly specific Tcell stimulation of a certain subpopulation of CTLs that possess ahigh-avidity TCR to the tumor antigen, stimulating and expanding them exvivo, and then introducing them into the patient. Adoptive immunotherapyis particularly effective if native lymphocytes are removed from thepatient before the infusion of tumor-specific cells. The idea behindthis type of therapy is that if the introduced high-avidity CTLs aresuccessful, once the tumor has been cleared, some of these cells willbecome memory T cells and will persist in the patient in case the cancerreappears. In 2002, a study was completed demonstrating regression ofmetastatic melanoma in patients that were treated under a regime ofadoptive immunotherapy following immunodepletion with cyclophosphamideand fludarabine (Dudley, et al. (2002) Science 298(5594): 850-854).Response rate was even higher if adoptive immunotherapy was preceded bytotal body irradiation (Dudley, et al. (2008) J Clin Oncol.26(32):5233-9).

However, adoptive immunotherapy has not been successful when the T cellsof interest containing high avidity TCRs cannot be readily expanded. Inaddition, it is often difficult to identify and isolate T cells withtherapeutic value from cancer patients because tumor antigens are oftenself-antigens, against which the patient's immune system is madetolerant through mechanisms of deletion or anergy of those T cell cloneswith the highest avidity. Thus, transfer of genes encoding high avidityTCRs into patient derived T cells has been proposed and demonstrated(see Rubenstein, et al. (2003) J of Immunology 170:1209-1217). Morerecently, using a mouse model of malignant melanoma, a statisticallysignificant decrease in tumor mass was found following introduction ofnormal lymphocytes that had been transduced with retroviral vectorscarrying human TCR genes specific for the gp-100 melanoma antigen (Abad,et al. (2008) J Immunother. 31(1):1-6) TCR gene therapy is alsodescribed in Morgan, et al. (2006) Science 314(5796):126-129 and Burns,et al. (2009) Blood 114(14):2888-2899.

However, transfer of any TCR transgenes into host T cells carries withit the caveats associated with most gene transfer methods, namely,unregulated and unpredictable insertion of the TCR transgene expressioncassette into the genome, often at a low level. Such poorly controlledinsertion of the desired transgene can result in effects of thetransgene on surrounding genes as well as silencing of the transgene dueto effects from the neighboring genes. In addition, the endogenous TCRgenes that are co-expressed in the T cell engineered with the introducedTCR transgene could cause undesired stimulation of the T cell by theantigen recognized by the endogenous TCR, undesired stimulation of the Tcell by unintended antigens due to the mispairing of the TCR transgenewith the endogenous TCR subunits creating a novel TCR complex with novelrecognition properties, or can lead to suboptimal stimulation againstthe antigen of interest by the creation of inactive TCRs due toheterodimerization of the transgene encoded TCR subunits with theendogenous TCR proteins. In fact, the risk of severe autoimmune toxicityresulting from the formation of self-reactive TCR from mispairing ofendogenous and exogenous chains has been recently highlighted in amurine model (Bendle, et al. (2010) Nature Medicine 16:565-570) and inhuman cells (van Loenen, et al. (2010) Proc Natl Acad Sci USA107:10972-10977). Additionally, the tumor-specific TCR may be expressedat suboptimal levels on the cell surface, due to competition with theendogenous and mispaired TCR for the CD3 molecules, required to expressthe complex on the cell surface.

Wilms tumor antigen (WT1 antigen) is a transcription factor normallyexpressed in embryonic cells. After birth, its expression is limited toonly a few cell types including hematopoietic stem cells. However, ithas been found to be overexpressed in many types of leukemias and solidtumors (see Inoue, et al. (1997) Blood 89:1405-1412) and may contributeto a lack of growth control in these cells. Due to the low expression ofWT1 in normal tissues, its expression on cancer cells makes it anattractive target for T-cell mediated therapy. TCR variants withincreased avidity to WT1 containing a modified cysteine to discouragemispairing between the endogenous TCR subunits and the transgene TCRshave been transduced into primary T cells and tested for functionality(Kuball, et al. (2007) Blood 109(6):2331-2338). The data demonstratedthat while T cells that had been freshly transduced with the WT1-TCRvariants had an increased antigen response as compared to thosetransduced with a wildtype TCR domain, after several rounds ofstimulation with the WT1 antigen, this improved antigen responsivenesswas lost (see Thomas, et al. (2007) J of Immunol 179 (9):5803-5810). Itwas concluded that even with the transgene-specific cysteinemodification, mispairing with the endogenous TCR peptides may play arole in reducing anti-WT1 avidity seen in cells transduced with theWT1-specific TCRs.

Thus, there remains a need for compositions that can introduce desiredTCR transgenes into a known chromosomal locus. In addition, there is aneed for methods and compositions that can selectively knock outendogenous TCR genes.

SUMMARY

Disclosed herein are compositions and methods for partial or completeinactivation or disruption of a TCR gene and compositions and methodsfor introducing and expressing to desired levels of exogenous TCRtransgenes into T lymphocytes, after or simultaneously with thedisruption of the endogenous TCR.

In one aspect, provided herein are zinc finger nucleases (ZFNs) thatcleave a TCR gene. In certain embodiments, the ZFNs bind to target sitesin a human TCR α gene and/or target sites in a human TCR β gene. In someembodiments, cleavage within the TCR gene(s) with these nucleasesresults in permanent disruption (e.g., mutation) of the TCR α and/or βgene(s). The zinc finger proteins may include 1, 2, 3, 4, 5, 6 or morezinc fingers, each zinc finger having a recognition helix that binds toa target subsite in the target gene. In certain embodiments, the zincfinger proteins comprise 4 or 5 or 6 fingers (designated F1, F2, F3, F4,F5 and F6 and ordered F1 to F4 or F5 or F6 from N-terminus toC-terminus) and the fingers comprise the amino acid sequence of therecognition regions shown in Table 5 and Table 6 and/or recognize thetarget sites shown in Tables 5 and 6.

Any of the proteins described herein may further comprise a cleavagedomain and/or a cleavage half-domain (e.g., a wild-type or engineeredFokI cleavage half-domain). Thus, in any of the ZFNs described herein,the nuclease domain may comprise a wild-type nuclease domain or nucleasehalf-domain (e.g., a FokI cleavage half domain). In other embodiments,the ZFNs comprise engineered nuclease domains or half-domains, forexample engineered FokI cleavage half domains that form obligateheterodimers. See, e.g., U.S. Patent Publication No. 2008/0131962.

In another aspect, the disclosure provides a polynucleotide encoding anyof the proteins described herein. Any of the polynucleotides describedherein may also comprise sequences (donor or patch sequences) fortargeted insertion into the TCR a and/or the TCR β gene. In yet anotheraspect, a gene delivery vector comprising any of the polynucleotidesdescribed herein is provided. In certain embodiments, the vector is anadenoviral vector (e.g., an Ad5/F35 vector) or a lentiviral vector (LV)including integration competent or integration-defective lentiviralvectors. Thus, also provided herein are adenoviral (Ad) vectors or LVscomprising a sequence encoding at least one zinc finger nuclease (ZFN)and/or a donor sequence for targeted integration into a target gene. Incertain embodiments, the Ad vector is a chimeric Ad vector, for examplean Ad5/F35 vector. In certain embodiments, the lentiviral vector is anintegrase-defective lentiviral vector (IDLV) or an integration competentlentiviral vector. In certain embodiments the vector is pseudo-typedwith a VSV-G envelope, or with other envelopes. In additionalembodiments, the target gene is the human TCR α gene. In certainembodiments, the target gene is the human TCR β gene. The vectorsdescribed herein may also comprise donor sequences. In additionalembodiments, the donor sequences comprise human TCR genes that arespecific for an MHC/antigen complex of interest. In some embodiments,the donor sequences may comprise the human TCR α and/or the human TCR βgenes that are specific for an MHC/antigen complex of interest. Incertain embodiments, a single vector comprises sequences encoding one ormore ZFNs and the donor sequence(s). In other embodiments, the donorsequence(s) are contained in a first vector and the ZFN-encodingsequences are present in a second vector. In further embodiments, theZFN-encoding sequences are present in a first vector and the TCR a geneof interest is present in a second vector and the TCR β gene of interestis present in a third vector. In some embodiments, the TCR genes ofinterest are inserted into the location of the endogenous TCR genes, andin other embodiments the TCR genes of interest are inserted intorandomly selected loci, or into a separate locus after genome-widedelivery. In some embodiments, the separate locus for TCR transgeneinsertion is the PPP1R12C locus (also known as AAVS1, see U.S PatentPublication Number 20080299580). In other embodiments, the TCR transgeneis inserted into a CCR-5 locus.

In yet another aspect, the disclosure provides an isolated cellcomprising any of the proteins, polynucleotides and/or vectors describedherein. In certain embodiments, the cell is selected from the groupconsisting of a stem/progenitor cell, a T-cell (e.g., CD4⁺ T-cell). In astill further aspect, the disclosure provides a cell or cell line whichis descended from a cell or line comprising any of the proteins,polynucleotides and/or vectors described herein, namely a cell or cellline descended (e.g., in culture) from a cell in which TCR has beeninactivated by one or more ZFNs and/or in which a TCR-encoding donorpolynucleotide has been stably integrated into the genome of the cell.Thus, descendants of cells as described herein may not themselvescomprise the proteins, polynucleotides and/or vectors described herein,but, in these cells, a TCR gene is inactivated and/or a TCR-encodingdonor polynucleotide is integrated into the genome and/or expressed.

In another aspect, described herein are methods of inactivating a TCRgene in a cell by introducing one or more proteins, polynucleotidesand/or vectors into the cell as described herein. In any of the methodsdescribed herein the ZFNs may induce targeted mutagenesis, targeteddeletions of cellular DNA sequences, and/or facilitate targetedrecombination at a predetermined chromosomal locus. Thus, in certainembodiments, the ZFNs delete or insert one or more nucleotides of thetarget gene. In some embodiments the TCR gene is inactivated by ZFNcleavage followed by non-homologous end joining. In other embodiments, agenomic sequence in the target gene is replaced, for example using a ZFN(or vector encoding said ZFN) as described herein and a “donor” sequencethat is inserted into the gene following targeted cleavage with the ZFN.The donor sequence may be present in the ZFN vector, present in aseparate vector (e.g., Ad or LV vector) or, alternatively, may beintroduced into the cell using a different nucleic acid deliverymechanism.

In another aspect, methods of using the zinc finger proteins and fusionsthereof for mutating a TCR gene and/or inactivating TCR function in acell or cell line are provided. Thus, a method for inactivating a TCRgene in a human cell is provided, the method comprising administering tothe cell any of the proteins or polynucleotides described herein.

In yet another aspect, the disclosure provides a method for treating orpreventing cancer, infections, autoimmune disorders, and/orgraft-versus-host disease (GVHD) in a subject, the method comprising:(a) introducing, into a cell (e.g., lymphocyte, stem cell, progenitorcell, etc.), a first nucleic acid encoding a first polypeptide, whereinthe first polypeptide comprises: (i) a zinc finger DNA-binding domainthat is engineered to bind to a first target site in a TCR gene; and(ii) a cleavage domain; under conditions such that the polypeptide isexpressed in the cell, whereby the polypeptide binds to the target siteand cleaves the endogenous TCR gene; and (b) introducing, into the cell,a second nucleic acid encoding a second polypeptide, wherein the secondpolypeptide comprises: (i) a zinc finger DNA-binding domain that isengineered to bind to a second target site in a TCR gene; and (ii) acleavage domain; under conditions such that the polypeptide is expressedin the cell, whereby the polypeptide binds to the target site andcleaves the endogenous TCR gene; and (c) introducing into the cell athird nucleic acid comprising a nucleic acid encoding a TCR gene or TCRgenes, specific for a tumor specific antigen in an MHC complex, suchthat the third nucleic acid is introduced into the endogenous TCR geneand the cell with the introduced third nucleic acid treats or prevents.In certain embodiments, steps (a)-(c) are performed ex vivo and themethod further comprises, following step (c), the step of introducingthe cell into the subject. In certain embodiments, the third nucleicacid encoding the TCR gene(s) is expressed under the control ofbi-directional promoters (e.g., PGK, EF1α, etc.). In other embodiments,the TCR gene(s) are expressed from bicistronic cassettes (e.g., usingviral 2A peptides or an IRES sequence) or by multiple LVs expressingdifferent TCR genes under monodirectional promoters. In certainembodiments, the cell is selected from the group consisting of astem/progenitor cell, or a T-cell.

In any of the methods describes herein, the first nucleic acid mayfurther encode a second polypeptide, wherein the second polypeptidecomprises: (i) a zinc finger DNA-binding domain that is engineered tobind to a second target site in the TCR gene; and (ii) a cleavagedomain; such that the second polypeptide is expressed in the cell,whereby the first and second polypeptides bind to their respectivetarget sites and cleave the TCR gene.

In another aspect, the disclosure also provides a method for treating orpreventing cancer in a subject, the method comprising: (a) introducing,into a cell, a first nucleic acid encoding a first polypeptide, whereinthe first polypeptide comprises: (i) a zinc finger DNA-binding domainthat is engineered to bind to a first target site in a TCR gene; and(ii) a cleavage domain; under conditions such that the polypeptide isexpressed in the cell, whereby the polypeptide binds to the target siteand cleaves the endogenous TCR; and (b) introducing, into a cell, asecond nucleic acid encoding a second polypeptide, wherein the secondpolypeptide comprises: (i) a zinc finger DNA-binding domain that isengineered to bind to a first target site in a safe harbor locus (e.g.,PPP1R12C, CCR5); and (ii) a cleavage domain; under conditions such thatthe polypeptide is expressed in the cell, whereby the polypeptide bindsto the target site and cleaves in the safe harbor locus (e.g., PPP1R12C,CCR5) and (c) introducing into the cell a third nucleic acid comprisinga donor nucleic acid encoding a TCR gene or TCR genes specific for atumor specific antigen in an MHC complex; and (d) introducing the cellinto the subject. The nucleic acids comprising the TCR specific ZFN maybe introduced simultaneously with the ZFN specific for the safe-harborlocus and the donor nucleic acid molecule, or the nucleic acid encodingthe TCR-specific ZFN may be introduced into the cell in a first step,and then the safe harbor locus (e.g., PPP1R12C, CCR5)-specific ZFNs andthe donor nucleic acid molecule may be introduced in a second step.

The disclosure also provides a method of preventing or treating a cancerin a subject comprising introducing, into a subject, a viral deliveryparticle wherein the viral delivery particle comprises (a) a firstnucleic acid encoding a first polypeptide, wherein the first polypeptidecomprises: (i) a zinc finger DNA-binding domain that is engineered tobind to a first target site in a TCR gene; and (ii) a cleavage domain;under conditions such that the polypeptide is expressed in the cell,whereby the polypeptide binds to the target site and cleaves theendogenous TCR; and (b) a second nucleic acid encoding a secondpolypeptide, wherein the second polypeptide comprises: (i) a zinc fingerDNA-binding domain that is engineered to bind to a first target site ina safe harbor locus (e.g., PPP1R12C, CCR5); and (ii) a cleavage domain;under conditions such that the polypeptide is expressed in the cell,whereby the polypeptide binds to the target site and cleaves the safeharbor locus (e.g., PPP1R12C, CCR5); and (c) a third nucleic acidencoding a third polypeptide, wherein the third polypeptide comprises:(i) a zinc finger DNA-binding domain that is engineered to bind to asecond target site in a safe harbor locus (e.g., PPP1R12C, CCR5); and(ii) a cleavage domain; under conditions such that the polypeptide isexpressed in the cell, whereby the polypeptide binds to the target siteand cleaves at the safe harbor locus (e.g., PPP1R12C, CCR5); and (d) athird nucleic acid comprising a donor nucleic acid encoding a TCR geneor TCR genes specific for a tumor specific antigen in an MHC complex;such that the endogenous TCR gene is cleaved and rendered inactive, andthe safe harbor gene (e.g., PPP1R12C, CCR5) is cleaved and the TCR genespecific for a tumor specific antigen in an MHC complex becomes insertedinto the endogenous TCR gene. In certain embodiments, the method furthercomprises, following step (d), the step of introducing the cell into thesubject.

In any of the methods described herein, a viral delivery particle can beused to deliver one or more of the polynucleotides (ZFN-encoding and/ordonor polynucleotides). Furthermore, in any of the methods andcompositions described herein, the cell can be, for example, astem/progenitor cell (e.g., a CD34⁺ cell), or a T-cell (e.g., a CD4⁺cell).

Furthermore, any of the methods described herein can be practiced invitro, in vivo and/or ex vivo. In certain embodiments, the methods arepracticed ex vivo, for example to modify PBMCs, e.g., T-cells, to makethem specific for a tumor antigen/MHC complex of interest to treat atumor in a subject. Non-limiting examples of cancers that can be treatedand/or prevented include lung carcinomas, pancreatic cancers, livercancers, bone cancers, breast cancers, colorectal cancers, leukemias,ovarian cancers, lymphomas, brain cancers and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B, depict construction and expression of a Wilms tumorantigen (WT1) specific lentiviral vector. FIG. 1A depicts a diagram ofthe genes encoding a codon-optimized, cysteine-modified TCR specific foran HLA-A2-restricted peptide from the Wilms tumor antigen 1 (WT1) clonedinto a third generation lentiviral vector (LV) under the control of abi-directional PGK or EF1α promoter. See, Amendola, et al. (2005) NatureBiotechnology 23(1):108-116 and U.S. Patent Publication No. 2006/200869.FIG. 1B is a graph depicting a time course of Vβ21 TCR expression inlentivirus transduced CD8⁺ cells cultured in the presence of 5 ng/ml ofIL7 and IL15. Vβ21 relative fluorescence intensity (RFI) was calculatedas the ratio of the mean fluorescence intensity (MFI) of Vβ21 measuredin PGK-WT1 (open squares) or EF1α-WT1 (“X”) genetically modifiedlymphocytes/the MFI of Vβ21 measured in T cells naturally expressingVβ21.

FIGS. 2A through 2C, are graphs depicting results of cells transducedwith TCR constructs. FIG. 2A depicts induction of γIFN production bystimulation of the cells with WT1+/HLA A2-K562 cells (the indicatedprimary AML or K562 cells (right most bar in graph)) transduced withvectors expressing the transgenic TCRs either from the PGK/mCMV dualpromoter combination (left side group of 4 bars) or the EF1α/mCMV dualpromoter (right side group of 4 bars) following exposure to WT1+HLA-A2+or WT1+HLA-A2− (negative control) primary leukemic blasts from AMLpatients (designated as AML1 (left most bar), AML2 (second bar from theleft) and AML3 (third bar from the left)). FIGS. 2B and 2C demonstratethe percent killing of the leukemic blasts from AML1 and AML2 (solidlines, closed circles) by the TCR modified cells. Dotted lines representresidual killing of the leukemic blasts by the TCR modified cells in thepresence of an excess of cold (not labeled) HLA-A2 target cells, loadedwith the WT1 proper peptide.

FIGS. 3A and 3B, are graphs depicting GFP expression followingintroduction of ZFNs targeted to a safe harbor locus together with a GFPdonor. FIGS. 3A and 3B demonstrate the increase in the percentage of GFPpositive cells in relation to the amount of Ad5/F35 CCR5-specific ZFN(FIG. 3A) or Ad5/F35 AAVS1-specific ZFN (FIG. 3B) and -IDLV GFP donorDNA cassette used.

FIGS. 4A and 4B, depict diagrams of exemplary TCR-α and TCR-β donormolecules (FIG. 4A) and the TCR-β genes (FIG. 4B). FIG. 4A depicts acassette containing WT1-specific TCR-α and TCR-β donor molecules andshows the regions of homology to the CCR5 integration site. FIG. 4Bdepicts the genomic arrangement of the two TCR-β constant regions inK562 cells (TRBC1 and TRBC2).

FIG. 5 depicts the percent modification for several pairs ofTCR-β-specific ZFNs in K562 cells as measured by a Cel-I Surveyor™mismatch assay (“Cel-I assay” Transgenomic) The cells were incubatedinitially at 30° C. following transfection, with either 0.1 or 0.4 μg ofZFN plasmid. Percent modification is shown at the bottom of the lanes.

FIGS. 6A and 6B, depict percent modification for TCR-α specific ZFNs inK562 cells as measured by a Cel-I assay. FIG. 6A depicts the resultsfrom a Cel-I assay on cells where the ZFNs were targeted to Exon 1. FIG.6B depicts the results of a Cel I mismatch assay where the ZFNs weretargeted to Exon 3. “GFP” indicates cells transduced with GFP onlyvectors. Percent alteration (NHEJ) is indicated at the bottom of thelanes.

FIGS. 7A through 7F, depict ZFN-mediated cleavage of TCR-β. Untransducedand transduced Jurkat cells using the TCR-β specific ZFN pair 16783 and16787 at two concentrations of vector demonstrated the loss of CD3signal at the cell surface (from 2.7% CD3(−) to 20.2% CD3(−) (seeExample 6). FIGS. 7A and 7B show the results of Cel-I assays at theTRBC1 (FIG. 7A) and the TRBC2 (FIG. 7B) locus in Jurkat cells anddemonstrate that cleavage has occurred. The measured % gene modificationis indicated at the bottom of each lane. FIG. 7C is a graph depictingthat sorted CD3− primary human lymphocytes can survive in the presenceof IL7 and TL15. “UT” indicates untreated cells. FIG. 7D shows thepercent modification (NHEJ), as assayed by the Cel-I assay observed inthe primary T-cell cell pools treated with TCR-beta specific ZFNs.“Bulk” indicates percent of NHEJ observed for the ZFN treated cell pool,while CD3+ or CD3− shows the NHEJ observed for cells that sorted eitheras CD3+ or CD3−. “UT” indicates cells that were not treated. The percentNHEJ detected by the assay is indicated at the bottom of the lanes. FIG.7E is a graph depicting percent CD3− cells and demonstrates thepersistence of CD3− cells over time (percent of CD3− cells stays fairlyconstant even up to 45 days) in cells treated with increasingconcentrations of ZFNs. FIG. 7F is a graph depicting that CD3− cellshave lost CD3 functionality since they do not appear to divide inresponse to non-specific mitogens.

FIG. 8 depicts the experimental outline and the FACS results for editingof the TCR-β locus in primary T lymphocytes and the re-introduction of aspecific TCR transgene. Cells used were either untreated primary T cellslymphocytes or lymphocytes pre-treated with the TCR-β-specific ZFNscarried by an IDLV and then sorted for CD3(−) primary T cells. Genetransfer was achieved after stimulation of T cells with cell-sized beadscoated with antibodies directed to CD3 and to CD28, and cell culture inthe presence of IL7 (5 ng/ml) and TL15 (5 ng/ml) to facilitate thegeneration of genetically modified central memory lymphocytes, accordingto European Patent Publication No EP1956080. As shown, cells that weresorted for being CD3(−) after treatment with the TCR-β specific ZFNs andthen have the WT1-TCRβ V21.3 transgene randomly integrated into thegenome using a lentiviral vector, show an increase in staining for bothCD3 and for V21.3, indicating primary T lymphocytes can undergoendogenous TCR disruption via NHEJ using the TCR-β-specific ZFNs andthen be re-targeted to recognize a specific antigen via the introductionof a new TCR encoded by a transgene cassette (PGK-WT1). As a control, UTcells also had the PGK-WT1 cassette inserted and showed a smallerpercentage of cells expressing Vβ 21.3 (26%) as compared to theZFN-treated, CD3(−) population (46%, 92% after polyclonal stimulation),indicating the disruption of the endogenous TCR may improve thecell-surface expression and functionality of the TCR expressed from thetransgene.

FIGS. 9A through 9C, depict expression of Vβ21 TCR. FIG. 9A depicts Vβ21TCR expression (upper histogram) and WT1₁₂₆₋₁₃₄ pentamer binding (lowerhistogram) in CD8⁺ TCR β chain disrupted and WT1 transduced cells thathave been sorted for CD3+ signal (TCR-p-edited), unedited WT1 LVtransduced cells (TCR-transferred), and untransduced lymphocytes treatedwith the same culture conditions. FIG. 9B shows a time course of surfaceexpression of Vβ21 TCR. Average+SD (n=2) of Vβ21 RFI is represented. RFIis calculated from the ratio of the MFI of Vβ21 measured in CD8⁺TCR-edited (open triangle) or TCR-transferred (dark circle)lymphocytes/the MFI of Vβ21 measured in CD8⁺ T cells naturallyexpressing Vβ21. FIG. 9C depicts the results of a cytotoxicity assaywith TCR-edited and TCR-transferred cells. Functional activity ismeasured by a ⁵¹Chromium release assay for lysis of labeled T2 cellspulsed with increasing concentrations of the WT1₁₂₆₋₁₃₄ HLA-A2restricted peptide, or with the irrelevant CMV-derived pp65₄₉₅₋₅₀₃HLA-A2 restricted peptide (10 μM) as negative control, at anEffector/Target (E/T) ratio of 12. Results are represented as average+SDof % of lysis (**, p<0.01, *, p<0.05 with Mann-Whitney test, TCR-editedn=6, TCR-transferred n=4).

FIGS. 10A and 10B, depict the functional activity of WT1 TCR-positive Tcell clones as tested by γIFN ELISpot assay. Clones were exposed to T2cells pulsed with 10 nM of the WT1₁₂₆₋₁₃₄ HLA-A2 restricted peptide(10A), or to allogeneic PBMC (10B) at a stimulator/responder ratio of1:1. The number of specific spots (open triangles and dark circles)observed is shown on the y axis as number of spots produced in presenceof stimulators minus the number of spots produced by effectors alone.The results show that the TCR-β edited clones exhibit a higher degree ofHLA A haplotype specificity than the TCR transferred cells which containboth the endogenous and the exogenous TCR genes.

FIG. 11 depicts Vβ21 expression in ZFN-edited and unedited cells. CD3(−)cells sorted from TRBC-disrupted lymphocytes and unedited cells and weretransduced at increasing MOI by LV encoding the Vβ21 gene of theWT1-specific TCR and the ΔLNGFR gene (see diagram at the top of figureshowing dual expression of the Vβ21 gene and the ΔLNGFR gene).Transduction efficiency was assessed as % of ΔLNGFR^(pos) lymphocytesand is shown. Vβ21 expression was measured on ΔLNGFR^(pos) cells anddemonstrates that the transduced Vβ21 gene can be expressed and formactive CD3 complexes with the endogenous TRAC genes. The meanfluorescence intensity (MFI) of Vβ21 is shown.

FIGS. 12A through 12C, depict CD3 expression in primary lymphocytestreated with ZFN targeting TCR α genes. FIG. 12A depicts a diagram ofthe human locus encoding the TCR alpha, total length 18 kb; TRAV,variable region genes, TRAD, diversity region genes, TRAC, constantregion gene. Displayed above the scheme of the locus are the genomic DNAsequences in TRAC targeted by each TRAC-ZFN. FIG. 12A discloses theprotein sequence as SEQ ID NO: 109 and the DNA sequence of SEQ ID NO:108. FIG. 12B depicts down-regulation of cell surface CD3 expressionmeasured by flow cytometry in primary human lymphocytes stimulated withbaCD3/CD28, cultured with 5 ng/ml IL-7, 5 ng/ml IL-15, and exposed toTRAC-ZFN IDLVs. The percent of CD3(−) cells is plotted. UT, Untransducedcells. Sorted CD3(−) cells were transduced with WT1-α OFP-LV resultingin expression of CD3 on transduced lymphocytes. FIG. 12C depicts a gelshowing the level of targeted gene disruption measured by the Cel-Iassay in primary lymphocytes exposed to TRAC-ZFN. The higher migratingproduct indicating wild type (w/t) gene is shown. Lower migratingproducts (NHEJ) indicate ZFN-directed gene disruption. “UT” refers tountransduced cells.

FIG. 13 depicts partial sequence of the genomic TRAC ZFN target site inZFN-treated human lymphocytes was amplified, cloned and sequenced toconfirm ZFN-induced modification. Sequence alignment revealed severalZFN-induced deletions and insertions (indels) within the target region.The left column indicates the number of clones retrieved while the rightcolumn indicates the number of deleted or inserted nucleotides. FIG. 13discloses SEQ ID NOS 110-143, respectively, in order of appearance.

FIG. 14 depicts expression of CD3 in following ZFN editing. Upper panelsshows results of studies in which activated T lymphocytes were treatedwith TRAC-ZFN-AdV (MOI 1000), and CD3(−) lymphocytes were sorted andtransduced with 3 μg-p24/10⁶ cells of PGK-WT1-α LV and CD3(+) cells weresorted. Surface expression of CD3 in transduced cells is shown. Afterone cycle of polyclonal stimulation, α-edited lymphocytes were treatedwith TRBC-ZFN-AdV (MOI 10⁴) and CD3(−) cells sorted and transduced with3 μg-p24/10⁶ cells of PGK-WT1-β LV. Surface expression of Vβ21 TCR andCD3 is shown on transduced cells before and after one cycle ofpolyclonal stimulation. Percent of events measured in each quadrant areshown, and the experimental timeline is shown on the bottom. Timeline isshown on the bottom.

FIG. 15 depicts Vβ21 TCR expression (upper histogram) and WT1₁₂₆₋₁₃₄pentamer binding (lower histogram) are shown in CD8(+) T cells with TCRα/β chains disrupted via introduction of ZFNs and sorting for CD3− cellsfollowed by transduction with the WT1 TCR chains and sorting for CD3+cells (TCR-edited), unedited WT1 LV transduced cells (TCR-transferred),and untransduced lymphocytes treated with the same culture conditions.The data show that the TCR edited cells have a higher level of Vβ21expression than those clones wherein both the endogenous and theexogenous TCR genes are present. It also demonstrates that the TCRedited cells display higher binding to the WT1 peptide than those cellsthat have both sets of TCR genes.

FIGS. 16A through 16C, are graphs depicting functional activity ofgenetically modified lymphocytes was tested by the γIFN ELISpot assay.Three weeks after polyclonal stimulation, TCR-α/β-edited and TCRtransferred lymphocytes were exposed to either i) T2 cells pulsed withincreasing concentrations of the WT1₁₂₆₋₁₃₄ HLA-A2 restricted peptide,or with the irrelevant CMV-derived pp65₄₉₅₋₅₀₃ HLA-A2 peptide (see FIG.16A, right side of figure) or ii) WT1⁺ HLA-A2(+) (black in FIG. 16B) orHLA-A2(−) (grey) leukemic cells harvested from AML patients with (dashedsymbols) or without (full symbols) pulsing with WT1₁₂₆₋₁₃₄ peptide (50nM). FIG. 16C shows similar results where allogenic PBMC were used astarget. All assays were performed at a stimulator/responder ratio of1:1. The number of specific spots is shown on the y axis as the numberof spots produced in the presence of stimulators minus the number ofspots produced by effectors alone. *=p<0.05, **=p<0.01, ***=p<0.001.

FIG. 17 depicts analysis for off-target cleavage by TRAC-specific ZFNs.The 15 most likely potential off target sites for the TRAC-specific ZFNs(identified by in silicio analysis) were analyzed following ZFNtreatment for cleavage by the Cel-I mismatch assay. Each potential offtarget site was analyzed in 5 samples: untransduced samples (UT),samples that were TRAC negative following TRAC-specific ZFN treatmentand sorting (TRAC neg), cells that were TRAC and TRBC negative followingsequential treatment with TRAC-specific ZFNs, the TRAC transgene andTRBC-specific ZFNs with sequential rounds of sorting (Double Neg), cellsthat were negative for the endogenous TRAC and TRBC loci following ZFNtreatment as well as modified to comprise non-wild type TRAC and TRBCtransgenes (Complete Edited), or TRBC negative following treatment withTRBC-specific ZFNs alone and sorting (TRBC Neg). Potential off targetsites are as labeled in Table 13.

FIG. 18 depicts analysis for off-target cleavage by TRBC-specific ZFNs.15 potential off target sites for the TRBC-specific ZNS (identified byin silicio analysis) were analyzed following ZFN treatment for cleavageby the Cel-I mismatch assay. Each potential off target site was analyzedin 5 samples: untransduced samples (UT), samples that were TRAC negativefollowing TRAC-specific ZFN treatment and sorting (TRAC neg), cells thatwere TRAC and TRBC negative following sequential treatment withTRAC-specific ZFNs, the TRAC transgene and TRBC-specific ZFNs withsequential rounds of sorting (Double Neg), cells that were negative forthe endogenous TRAC and TRBC loci following ZFN treatment as well asmodified to comprise non-wild type TRAC and TRBC transgenes (CompleteEdited), or TRBC negative following treatment with TRBC-specific ZFNsalone and sorting (TRBC Neg). Off target sites are as labeled in Table14. TRBC depicts modification of the intended target site in thesesamples.

DETAILED DESCRIPTION

Disclosed herein are zinc finger nucleases (ZFNs) targeting a TCR gene(TCR-ZFNs). These ZFNs efficiently generate a double strand break (DSB),for example at a predetermined site in a TCR coding region. ZFN-mediatedintroduction of a site-specific double strand break (DSB) in genes thatencode for the TCR gene can result in the specific and permanentdisruption of the endogenous TCR complex in human cells, including humanT cells. These cells can be selected from a pool by selecting for CD3(−)cells, and culturing them on IL7 and IL15. In addition, disclosed hereinare methods and compositions for the replacement of the endogenous TCRgenes with TCR transgenes of one's choice, either via random integrationor by site directed targeted integration.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see alsoInternational Patent Publication Nos. WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536; and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; InternationalPatent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO 02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman (1981) Advances in Applied Mathematics2:482-489. This algorithm can be applied to amino acid sequences byusing the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, (1986) Nucl. Acids Res. 14(6):6745-6763. An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, WI) in the“BestFit” utility application. The default parameters for this methodare described in the Wisconsin Sequence Analysis Package Program Manual,Version 8 (1995) (available from Genetics Computer Group, Madison, WI).A preferred method of establishing percent identity in the context ofthe present disclosure is to use the MPSRCH package of programscopyrighted by the University of Edinburgh, developed by John F. Collinsand Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (MountainView, CA). From this suite of packages the Smith-Waterman algorithm canbe employed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects sequenceidentity. Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at thefollowing internet address: http://www.ncbi.nlm.gov/cgi-bin/BLAST. Withrespect to sequences described herein, the range of desired degrees ofsequence identity is approximately 80% to 100% and any integer valuetherebetween. Typically the percent identities between sequences are atleast 70-75%, preferably 80-82%, more preferably 85-90%, even morepreferably 92%, still more preferably 95%, and most preferably 98%sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, DC; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, DC; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474; 2006/0188987; and2008/0131962 and U.S. Pat. Nos. 7,914,796; 8,034,598; and 8,771,986,incorporated herein by reference in their entireties.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

A “safe harbor locus” is a location within a genome that can be used forintegrating exogenous nucleic acids. The addition of exogenous nucleicacids into these safe harbor loci does not cause any significant effecton the growth of the host cell by the addition of the DNA alone.Non-limiting examples of safe harbor loci include the PPP1R12C locus(also known as AAVS1, see U.S Patent Publication No. 2008/0299580) andthe CCR5 locus (see U.S. Patent Publication No. 2008/0159996).

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression.

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel, et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields, et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and International PatentPublication No. WO 98/44350.

Nucleases

Described herein are nucleases (e.g., ZFNs or TAL nucleases) that can beused for inactivation of a TCR gene The nuclease may be naturallyoccurring or may be a chimera of a DNA-binding domain and a cleavagedomain. It will be apparent that within the chimera, the componentDNA-binding and cleavage domains may both be naturally occurring, mayboth be non-naturally occurring or one may be naturally occurring andthe other may be non-naturally occurring. Thus, any nuclease can be usedin the methods disclosed herein. For example, naturally-occurring homingendonucleases and meganucleases have very long recognition sequences,some of which are likely to be present, on a statistical basis, once ina human-sized genome. Exemplary homing endonucleases include I-SceI,I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI,I-SceIII, I-CreI, I-TevI, I-TevII and I-TevII. Their recognitionsequences are known. See also U.S. Pat. Nos. 5,420,032; 6,833,252;Belfort, et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon, et al.(1989) Gene 82:11-118; Perler, et al. (1994) Nucleic Acids Res.22:1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble, et al.(1996) J. Mol. Biol. 263:163-180; Argast, et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue.

It has also been reported that the specificity of homing endonucleasesand meganucleases can be engineered to bind non-natural target sites.See, for example, Chevalier, et al. (2002) Molec. Cell 10:895-905;Epinat, et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth, et al.(2006) Nature 441:656-659; Paques, et al. (2007) Current Gene Therapy7:49-66. Thus, any naturally occurring or engineered nuclease having aunique target site can be used in the methods described herein.

A. DNA-Binding Domains

The nucleases described herein typically include a DNA-binding domainand a cleavage domain. Any DNA-binding domain can be used in thepractice of the present invention.

In certain embodiments, zinc finger binding domains that are engineeredto bind to a sequence of choice are employed. See, for example, Beerli,et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann.Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol.19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637;Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. An engineeredzinc finger binding domain can have a novel binding specificity,compared to a naturally-occurring zinc finger protein. Engineeringmethods include, but are not limited to, rational design and varioustypes of selection. Rational design includes, for example, usingdatabases comprising triplet (or quadruplet) nucleotide sequences andindividual zinc finger amino acid sequences, in which each triplet orquadruplet nucleotide sequence is associated with one or more amino acidsequences of zinc fingers which bind the particular triplet orquadruplet sequence. See, for example, co-owned U.S. Pat. Nos. 6,453,242and 6,534,261, incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878; and WO 01/88197 and GB 2,338,237.

Enhancement of binding specificity for zinc finger binding domains hasbeen described, for example, in co-owned International PatentPublication No. WO 02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in related to U.S.Patent Publication Nos. 2003/0232410; 2005/0208489; 2005/064474;2005/0026157; 2006/0188987; and 2007/0134796; International PatentPublication No. WO 07/014275; and U.S. Pat. No. 7,972,854, thedisclosures of which are incorporated by reference in their entiretiesfor all purposes.

The zinc finger nucleases described herein bind in a TCR gene. Tables 5and 6(see Example 4) describes a number of zinc finger binding domainsthat have been engineered to bind to nucleotide sequences in the humanTCR gene. Each row describes a separate zinc finger DNA-binding domain.The DNA target sequence for each domain is shown in the first column(DNA target sites indicated in uppercase letters; non-contactednucleotides indicated in lowercase), and the second through fifthcolumns show the amino acid sequence of the recognition region (aminoacids −1 through +6, with respect to the start of the helix) of each ofthe zinc fingers (F1 through F4 or F5 or F6) in the protein. Alsoprovided in the first column is an identification number for eachprotein.

As described below, in certain embodiments, a four- or five-fingerbinding domain as shown in Tables 5 and 6 is fused to a cleavagehalf-domain, such as, for example, the cleavage domain of a Type IIsrestriction endonuclease such as FokI. A pair of such zincfinger/nuclease half-domain fusions are used for targeted cleavage, asdisclosed, for example, in U.S. Pat. No. 7,888,121.

For targeted cleavage, the near edges of the binding sites can separatedby 5 or more nucleotide pairs, and each of the fusion proteins can bindto an opposite strand of the DNA target.

In addition, domains from these naturally occurring or engineerednucleases can also be isolated and used in various combinations. Forexample, the DNA-binding domain from a naturally occurring or engineeredhoming endonucleases or meganuclease can be fused to a heterologouscleavage domain or half domain (e.g., from another homing endonuclease,meganuclease or TypeIIS endonuclease). These fusion proteins can also beused in combination with zinc finger nucleases described above.

In some embodiments, the DNA binding domain is an engineered domain froma TAL effector protein derived from the plant pathogen Xanthomonas (seeBoch, et al. (2009) Science 326: 1509-1512 and Moscou and Bogdanove(2009) Science 326: 1501). See, also, U.S. Pat. No. 8,586,526.

The nucleases described herein can be targeted to any sequence in anyTCR genomic sequence.

B. Cleavage Domains

The nucleases also comprise a nuclease (cleavage domain, cleavagehalf-domain). The cleavage domain portion of the fusion proteinsdisclosed herein can be obtained from any endonuclease or exonuclease.Exemplary endonucleases from which a cleavage domain can be derivedinclude, but are not limited to, restriction endonucleases and homingendonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, MA; and Belfort, et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., S1Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn, et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150; and 5,487,994; as wellas Li, et al. (1992) Proc. Natl. Acad Sci. USA 89:4275-4279; Li, et al.(1993) Proc. Natl. Acad Sci. USA 90:2764-2768; Kim, et al. (1994a) Proc.Natl. Acad Sci. USA 91:883-887; Kim, et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite, et al. (1998) Proc. Natl. Acad Sci. USA95:10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPatent Publication No. WO 07/014275, incorporated herein in itsentirety. Additional restriction enzymes also contain separable bindingand cleavage domains, and these are contemplated by the presentdisclosure. See, for example, Roberts, et al. (2003) Nucleic Acids Res.31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987;2008/0131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of FokI are all targets for influencing dimerizationof the FokI cleavage half-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFokI and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in certain embodiments, the mutation at 490 replaces Glu (E) withLys (K); the mutation at 538 replaces Iso (I) with Lys (K); the mutationat 486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., Example1 of U.S. Patent Publication No. 2008/0131962, the disclosure of whichis incorporated by reference in its entirety for all purposes.

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (FokI) as described in U.S. PatentPublication Nos. 2005/0064474 and 2008/0131962.

The engineered cleavage half-domains described herein may be obligateheterodimer mutants in which aberrant cleavage is minimized orabolished. See, e.g., Example 1 of International Patent Publication No.WO 07/139898. In certain embodiments, the engineered cleavagehalf-domain comprises mutations at positions 486, 499 and 496 (numberedrelative to wild-type FokI), for instance mutations that replace thewild type Gln (Q) residue at position 486 with a Glu (E) residue, thewild type Iso (I) residue at position 499 with a Leu (L) residue and thewild-type Asn (N) residue at position 496 with an Asp (D) or Glu (E)residue (also referred to as a “ELD” and “ELE” domains, respectively).In other embodiments, the engineered cleavage half-domain comprisesmutations at positions 490, 538 and 537 (numbered relative to wild-typeFokI), for instance mutations that replace the wild type Glu (E) residueat position 490 with a Lys (K) residue, the wild type Iso (I) residue atposition 538 with a Lys (K) residue, and the wild-type His (H) residueat position 537 with a Lys (K) residue or a Arg (R) residue (alsoreferred to as “KKK” and “KKR” domains, respectively). In otherembodiments, the engineered cleavage half-domain comprises mutations atpositions 490 and 537 (numbered relative to wild-type FokI), forinstance mutations that replace the wild type Glu (E) residue atposition 490 with a Lys (K) residue and the wild-type His (H) residue atposition 537 with a Lys (K) residue or a Arg (R) residue (also referredto as “KIK” and “KIR” domains, respectively). (See U.S. PatentPublication No. 2011/0201055).

Alternatively, the FokI nuclease domain variant known as “Sharkey” maybe used (see Guo, et al. (2010) J. Mol. Biol.doi:10.1016/j.jmb.2010.04.060).

Delivery

The nuclease described herein may be delivered to a target cellcontaining a TCR gene by any suitable means. Methods of deliveringproteins comprising DNA-binding domains are described, for example, inU.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882;6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824,the disclosures of all of which are incorporated by reference herein intheir entireties.

Nucleases as described herein may also be delivered using vectorscontaining sequences encoding one or more nucleases. Any vector systemsmay be used including, but not limited to, plasmid vectors, retroviralvectors, lentiviral vectors, adenovirus vectors, poxvirus vectors;herpesvirus vectors and adeno-associated virus vectors, etc. See, also,U.S. Pat. Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539;7,013,219; and 7,163,824, incorporated by reference herein in theirentireties.

In certain embodiments, the vector is a lentiviral vector. A lentiviralvector, as used herein, is a vector which comprises at least onecomponent part derivable from a lentivirus. A detailed list oflentiviruses may be found in Coffin, et al. (1997) “Retroviruses” ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 758-763). Lentiviral vectors can be produced generally by methodswell known in the art. See, e.g., U.S. Pat. Nos. 5,994,136; 6,165,782;and 6,428,953. Preferably, the lentiviral vector is an integrasedeficient lentiviral vector (IDLV). See, e.g., U.S. Patent PublicationNo. 2009/0117617. IDLVs may be produced as described, for example usinglentivirus vectors that include one or more mutations in the nativelentivirus integrase gene, for instance as disclosed in Leavitt, et al.(1996) J. Virol. 70(2):721-728; Philippe, et al. (2006) Proc. Natl Acad.Sci USA 103(47):17684-17689; and International Patent Publication No. WO06/010834. In certain embodiments, the IDLV is an HIV lentiviral vectorcomprising a mutation at position 64 of the integrase protein (D64V), asdescribed in Leavitt, et al. (1996) J. Virol. 70(2):721-728.

In certain embodiments, the vector is an adenovirus vector. Non-limitingexamples of Ad vectors that can be used in the present applicationinclude recombinant (such as E1-deleted), conditionally replicationcompetent (such as oncolytic) and/or replication competent Ad vectorsderived from human or non-human serotypes (e.g., Ad5, Ad11, Ad35, orporcine adenovirus-3); and/or chimeric Ad vectors (such as Ad5/F35) ortropism-altered Ad vectors with engineered fiber (e.g., knob or shaft)proteins (such as peptide insertions within the HI loop of the knobprotein). Also useful are “gutless” Ad vectors, e.g., an Ad vector inwhich all adenovirus genes have been removed, to reduce immunogenicityand to increase the size of the DNA payload. This allows, for example,simultaneous delivery of sequences encoding ZFNs and a donor sequence.Such gutless vectors are especially useful when the donor sequencesinclude large transgenes to be integrated via targeted integration.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer, and they readily infect a number of differentcell types. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in cells that provide one or more of thedeleted gene functions in trans. For example, human 293 cells supply E1function. Ad vectors can transduce multiple types of tissues in vivo,including non-dividing, differentiated cells such as those found inliver, kidney and muscle. Conventional Ad vectors have a large carryingcapacity. An example of the use of an Ad vector in a clinical trialinvolved polynucleotide therapy for antitumor immunization withintramuscular injection (Sterman, et al. (1998) Hum. Gene Ther.7:1083-1089).

Additional examples of the use of adenovirus vectors for gene transferin clinical trials include Rosenecker, et al. (1996) Infection 24:15-10; Welsh, et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez, et al.(1997) Hum. Gene Ther. 5:597-613; Topf, et al. (1998) Gene Ther.5:507-513.

In certain embodiments, the Ad vector is a chimeric adenovirus vector,containing sequences from two or more different adenovirus genomes. Forexample, the Ad vector can be an Ad5/F35 vector. Ad5/F35 is created byreplacing one or more of the fiber protein genes (knob, shaft, tail,penton) of Ad5 with the corresponding fiber protein gene from a B groupadenovirus such as, for example, Ad35. The Ad5/F35 vector andcharacteristics of this vector are described, for example, in Ni, et al.(2005) “Evaluation of biodistribution and safety of adenovirus vectorscontaining group B fibers after intravenous injection into baboons,” HumGene Ther 16:664-677; Nilsson, et al. (2004) “Functionally distinctsubpopulations of cord blood CD34+ cells are transduced by adenoviralvectors with serotype 5 or 35 tropism,” Mol Ther 9:377-388; Nilsson, etal. (2004) “Development of an adenoviral vector system with adenovirusserotype 35 tropism; efficient transient gene transfer into primarymalignant hematopoietic cells,” J Gene Med 6:631-641; Schroers, et al.(2004) “Gene transfer into human T lymphocytes and natural killer cellsby Ad5/F35 chimeric adenoviral vectors,” Exp Hematol 32:536-546;Seshidhar, et al. (2003) “Development of adenovirus serotype 35 as agene transfer vector,” Virology 311:384-393; Shayakhmetov, et al. (2000)“Efficient gene transfer into human CD34(+) cells by a retargetedadenovirus vector,” J Virol 74:2567-2583; and Sova, et al. (2004), “Atumor-targeted and conditionally replicating oncolytic adenovirus vectorexpressing TRAIL for treatment of liver metastases,” Mol Ther 9:496-509.As noted above, ZFNs and polynucleotides encoding these ZFNs may bedelivered to any target cell. Generally, for inactivating a gene CCR-5,the cell is an immune cell, for example, a lymphocyte (B-cells, T-cellssuch as T helper (T_(H)) and T cytotoxic cells (T_(C)), null cells suchas natural killer (NK) cells); a mononuclear cell (monocytes,marcophages); a granulocytic cell (granulocytes, neutrophils,eosinophils, basophils); a mast cell; and/or a dendritic cell(Langerhans cells, interstitial dendritic cells, interdigitatingdendritic cells, circulating dendritic cells). Macrophages, Blymphocytes and dendritic cells are exemplary antigen-presenting cellsinvolved in T_(H) cell activation. In certain embodiments, the targetcell is a T_(H) cell, characterized by expression of CD4 on the surface.The target cell may also be a hematopoietic stem cell, which may giverise to any immune cell.

Applications

The disclosed methods and compositions can be used for inactivation of aTCR genomic sequence. As noted above, inactivation includes partial orcomplete repression of the endogenous TCR α and/or β gene expression ina cell. Inactivation of a TCR gene can be achieved, for example, by asingle cleavage event, by cleavage followed by non-homologous endjoining, by cleavage at two sites followed by joining so as to deletethe sequence between the two cleavage sites, by targeted recombinationof a missense or nonsense codon into the coding region, by targetedrecombination of an irrelevant sequence (i.e., a “stuffer” sequence) oranother coding sequence of interest into the gene or its regulatoryregion, so as to disrupt the gene or regulatory region, or by targetingrecombination of a splice acceptor sequence into an intron to causemis-splicing of the transcript. Inactivation of an endogenous TCR genecan also be accomplished by targeted recombination of a TCR gene(s)specific for a tumor antigen/MHC complex of interest.

There are a variety of applications for nuclease-mediated inactivation(knockout or knockdown) of a TCR gene. For example, the methods andcompositions described herein allow for the generation and/ormodification of cells lines (for therapeutic and non-therapeutic uses).Inactivation of the endogenous TCR gene(s) may be coupled with theinsertion of genes encoding high avidity TCRs or chimeric antigenreceptors (CARS, see Cartellieri, et al. (2010) J Biomed and Biotech,Vol 2010, Article ID 956304) against a known target, and the resultanttransgenic cells (or descendants of these cells having the samecharacteristics) may be used as cellular therapeutics. Alternatively,the re-targeting of the T cell may occur in vivo, using viral vectors todeliver both the genes encoding the TCR-specific nucleases and the highavidity TCR on a donor nucleic acid. In either case, the materials andmethods of the invention may be used in the treatment of cancer. Cellsmodified in vitro may also be used for modeling studies or for screeningto find other types of therapeutics that may also work in concert withthe TCR modification. Any type of cancer can be treated, including, butnot limited to lung carcinomas, pancreatic cancers, liver cancers, bonecancers, breast cancers, colorectal cancers, ovarian cancers, leukemias,lymphomas, brain cancers and the like. Other diseases that may betreated with the technology of the invention include fungal, bacterialand viral infections as well as autoimmune diseases andgraft-versus-host disease (GvHD).

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity and understanding,it will be apparent to those of skill in the art that various changesand modifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing disclosure andfollowing examples should not be construed as limiting.

EXAMPLES Example 1: Expression of an Optimized, High Affinity WT-1 TCRConstruct

Genes encoding for a codon-optimized, cysteine-modified TCR specific foran HLA-A2-restricted peptide from the Wilms tumor antigen 1 (WT1),specifically the WT1₁₂₆₋₁₃₄ peptide (Kuball, et al. (2007) Blood109(6):2331-8) and single α21 or β21 WT1 specific TCR chains were clonedinto bidirectional self-inactivating transfer vectorspCCLsin.PPT.ΔLNGFR.minCMV.WPGK.eGFP.Wpre orpCCLsin.cPPT.ΔLNGFR.min.CMV.hEF1a.eGFP.Wpre as described in Amendola, etal. (2005) Nature Biotechnology 23(1):108-116, Thomas, et al. (2007) J.Immunol 179 (9):5803-5810, and U.S. Patent Publication No. 2006/200869(see FIG. 1A)

The vectors were packaged using an integrase-competent third generationlentivirus vector system, and pseudotyped by VZV envelope, essentiallyas described in Follenzi and Naldini (2002) Methods in Enzymology346:454-465. The lentiviral vectors were then used to transduce cellsusing standard techniques (see below) and cells were characterized byFACs analysis to determine if the exogenous TCRs were being expressed onthe cell surface.

As shown below in Table 1, the WT-1 specific TCR construct was highlyexpressed, whether driven from the PGK/mCMV dual promoter combination orthe EF1α/mCMV dual promoter construct. Numbers in Table are presented aspercent of total signal present in the quadrant gated for VB21expression and WT1-HLA-A2 pentamer binding.

TABLE 1 Expression of WT-1 TCR Promoter Day 14 Day 22 PGK 12.1 21.3 EF1α1.48 5.16 Untransduced = 0.085

Transduction of T cells was accomplished by activating the cells withanti-CD3/anti-CD28 antibody-conjugated magnetic beads (Clin ExVivoCD3/CD28; Invitrogen) (baCD3/CD28) where the cells were cultured in IMDM(GIBCO-BRL), 10% FCS with low dose IL-7/IL-15 as described in EuropeanPatent Publication No. EP1956080 and Koncko, et al. (2009) Blood113:1006-1015. This procedure preserved an early T cell differentiationphenotype (CD45RA−/+CD62L+, CD28+CD27+, IL7Ra+, IL-2+ γIFN−/+), and thecells proliferated indistinguishably from untransduced lymphocytes. Inthese conditions, the PGK dual promoter proved to be superior to theEF1α dual promoter in sustaining stochiometric expression ofWT1-specific TCR chains, suggesting that the PGK bi-directional promoterexerts a higher activity in the antisense direction than thebi-directional EF1α promoter. Both promoters however, when tested in thecontext of a lentiviral vector, supported TCR expression at levelsappropriate for efficient HLA-A2/WT1 pentamer binding (16%), for >70days after initial stimulation (see FIG. 1 i ).

TCR transduced cells were also able to exhibit specific γIFN productionand cytotoxic activity against WT1+HLA-A2+ primary leukemic blasts fromAML patients. In particular, γIFN production in cells transduced withvectors expressing the transgenic TCRs either from the PGK/mCMV dualpromoter combination or the EF1α/mCMV dual promoter was increased (FIG.2A) as was % killing (lysis) by the TCR modified cells (FIGS. 2B and2C). In addition, γIFN production was inhibited in the editedlymphocytes (FIG. 2D), in the presence of unlabelled targets expressingthe HLA-restriction element and pulsed with the target peptide.

Example 2: Efficient Integration of a Transgene into the CCR5 Locus ofCentral Memory T Cells

To test the idea of integrating the WT-1 specific TCR genes into acentral memory T cell, GFP was used first as a donor nucleic acid tomonitor transduction efficiency and GFP expression from the site ofintegration. The CCR5 locus was chosen because it has been shown thatCCR5 knockout cells are fully functional (see U.S. Patent PublicationNo. 2008/0159996). In addition, the PPP1R12C (AAVS1) locus was similarlytargeted (see U.S. Patent Publication No. 2008/0299580) The GFP-encodingdonor was transduced into the cell using an IDLV vector and theCCR5-specific ZFNs or AAVS1-specific ZFNs were introduced using aAd5/F35 vector as described above. GFP expression was measured 20 daysfollowing transduction.

As shown in FIG. 3 , ZFN-mediated integration of GFP transgenes resultedin increased GFP signals, including in relation to the amount of Ad5/F35donor used (FIGS. 3A and 3B). Table 2 below shows the increase in thepercent of GFP positive cells in the presence of donor or donor plusZFNs.

TABLE 2 GFP signal, percent positive cells Insert site UT +donor donor +ZFN CCR5 0.038 0.083 6.11 AAVS1 0.015 0.18 4.38

Example 3: Integration of WT-1 Specific TCR Transgenes into the CCR5Locus of Jurkat TCR β-Negative Cells

The WT-1 specific TCR transgene construct was then used for targetedintegration into the CCR5 locus of Jurkat cells that are TCR β-negativefollowing treatment with TCR-β specific ZFNs. Cells were transfectedusing standard techniques with WT-1 TCR construct similar to thatdescribed in Example 1.

As seen in Table 4, after introduction of the WT-1 TCR donor (WT1-TCRIDLV) and the CCR5-specific ZFNs (Ad-ZFNs), there is a marked increasein Vβ21 staining or signal, while without the donor or the ZFNs, onlybackground Vβ21 signal is seen. Thus, ZFN-mediated integration of theWT-1 specific TCR into the CCR5 locus occurred in a substantialpercentage of the cells.

TABLE 4 Percent of total signal from VB21+ expression WT1-TCR IDLV + + +− Ad ZFNs + ++ − − Percent VB21+ 16.6 18.7 2.27 0.81

Example 4: Design of TCR-Specific ZFNs

TCR-specific ZFNs were constructed to enable site specific introductionof double strand breaks at either the TCRα and/or TCRβ genes. ZFNs weredesigned and incorporated into plasmids or IDLV vectors essentially asdescribed in Urnov, et al. (2005) Nature 435(7042):646-651, Lombardo, etal. (2007) Nat Biotechnol. November 25(11):1298-306, and U.S. PatentPublication No. 2008/0131962. The recognition helices for exemplary ZFNpairs as well as the target sequence are shown below in Tables 5 and 6.Target sites of the TCR zinc-finger designs are shown in the firstcolumn. Nucleotides in the target site that are contacted by the ZFPrecognition helices are indicated in uppercase letters; non-contactednucleotides indicated in lowercase.

TABLE 5 TCR-α Zinc-finger Designs ZFN Name Target sequence F1 F2 F3 F4F5 F6 25529  QSGDLTR QRTHLKA QSGDRNK DRSNLSR RSDA N/A (ex 1) (SEQ ID(SEQ ID (SEQ ID (SEQ ID LTQ ctATGGAC NO: 2) NO: 3) NO: 4) NO: 5) (SEQ tTCAAGAG ID CAacagtg NO:  ctgt 6) (SEQ ID NO: 1) 25528  TSGSLSR QSSVRNSRSDNLST DRSALAR LKQN N/A (ex 1) (SEQ ID (SEQ ID (SEQ ID (SEQ ID LDActCATGTC NO: 8) NO: 9) NO: 10) NO: 11) (SEQ  TAGcACAG ID TTttgtct NO: gtga 12) (SEQ ID NO: 7) 25535  DRSALSR QSGHLSR DRSDLSR RSDALSR DRSD N/A(ex 1) (SEQ ID (SEQ ID (SEQ ID (SEQ ID LSR gtGCTGTG NO: 14) NO: 15)NO: 16) NO: 17) (SEQ  GCCtGGAG ID CAacaaat NO:  ctga 16) (SEQ ID NO: 13)25534  DRSNLSR QKTSLQA DRSALSR QSGNLAR GKEE RSSD (ex 1) (SEQ ID (SEQ ID(SEQ ID (SEQ ID LNE LSR ttGCTCTT NO: 5) NO: 19) NO: 14) NO: 20) (SEQ (SEQ  GAAGTCcA ID ID TAGACctc NO:  NO: atgt 21) 22) (SEQ ID NO: 18)25537  GNVDLIE RSSNLSR RSDALSV DSSHRTR WRSC N/A (ex 1) (SEQ ID (SEQ ID(SEQ ID (SEQ ID RSA gcTGTGGC NO: 24) NO: 25) NO: 26) NO: 27) (SEQ CTGGAGCA ID Acaaatct NO:  gact 28) (SEQ ID NO: 23) 25536  DSSDRKKRSDNLSV RRFILRG QSGDLTR TSGS N/A (ex 1) (SEQ ID (SEQ ID (SEQ ID (SEQ IDLTR ctGTTGCT NO: 30) NO: 31) NO: 32) NO: 2) (SEQ  cTTGAAGT ID CCatagacNO:  ctca 33) (SEQ ID NO: 29) 25538  QSGDLTR QTSTLSK QSGHLSR DRSDLSRRSDA N/A (ex 1) (SEQ ID (SEQ ID (SEQ ID (SEQ ID LAR ctGTGGCC NO: 2)NO: 35) NO: 15) NO: 16) (SEQ  tGGAGCAA ID CAaatctg NO:  actt 36) (SEQ IDNO: 34) 25540  QSGDLTR WRSSLAS QSGDLTR HKWVLRQ DRSN N/A (ex 1) (SEQ ID(SEQ ID (SEQ ID (SEQ ID LTR ctGACTTT NO: 2) NO: 38) NO: 2) NO: 39) (SEQ GCATGTGC ID Aaacgcct NO:  tcaa 40) (SEQ ID NO: 37) 25539  QSGDLTRQWGTRYR ERGTLAR RSDNLRE QSGD TSGS (ex 1) (SEQ ID (SEQ ID (SEQ ID (SEQ IDLTR LTR ttGTTGCT NO: 2) NO: 42) NO: 43) NO: 44) (SEQ  (SEQ  cCAGGCCA IDID  CAGCActg NO:  NO: ttgc 2) 33) (SEQ ID NO: 41) 22199  RSAHLSR DRSDLSRRSDHLSV QNNHRIT N/A N/A (ex 3) (SEQ ID (SEQ ID (SEQ ID (SEQ ID tgAAAGTGNO: 46) NO: 16) NO: 47) NO: 48) GCCGGGtt taatctgc tcat (SEQ ID NO: 45)22189  QRSNLVR RNDDRKK TSGNLTR TSANLSR N/A N/A (ex 3) (SEQ ID (SEQ ID(SEQ ID (SEQ ID agGAGGAT NO: 50) NO: 51) NO: 52) NO: 53) TCGGAAcccaatcact gaca (SEQ ID NO: 49) 25572  DRSTLRQ QRSNLVR RNDDRKK RSAHLSRQSGH N/A (ex 3) (SEQ ID (SEQ ID (SEQ ID (SEQ ID LSR gaGGAGGA NO: 55)NO: 50) NO: 51) NO: 46) (SEQ  tTCGGAAC ID CCaatcac NO:  tgac 15) (SEQ IDNO: 54) 25573  QRSNLVR RNDDRKK QSGHLAR QSGHLSR N/A N/A (ex 3) (SEQ ID(SEQ ID (SEQ ID (SEQ ID gaGGAGGA NO: 50) NO: 51) NO: 56) NO: 15)tTCGGAAc ccaatcac tgac (SEQ ID NO: 54) 22199  RSAHLSR DRSDLSR RSDHLSVQNNHRIT N/A N/A (ex 3) (SEQ ID (SEQ ID (SEQ ID (SEQ ID tgAAAGTG NO: 46)NO: 16) NO: 47) NO: 48) GCCGGGtt taatctgc tcat (SEQ ID NO: 57)

TABLE 6 TCR-β Zinc-finger Designs ZFN  Name Tar- get se- quence F1 F2 F3F4 F5 F6 16783 RSDVLSA DRSNRIK RSDVLSE QSGNLAR QSGS N/A ccGTAGA (SEQ ID(SEQ ID (SEQ ID (SEQ ID LTR  ACTGGAC NO: 59) NO: 60) NO: 61) NO: 20)(SEQ TTGacag ID cggaagt NO: (SEQ ID 62) NO: 58) 16787 RSDHLST RSDNLTRDRSNLSR TSSNRKT RSAN RNDD tcTCGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID LAR RKK GAATGAC NO: 64) NO: 65) NO: 5) NO: 66) (SEQ (SEQ  GAGTGGa ID  IDcccagga NO: NO: (SEQ ID 67) 51) NO: 63) 22409 RSDHLST RSDNLTR DRSNLSRLQFNRNQ RSAN RNDD tcTCGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID LAR RKKGAATGAC NO: 64) NO: 65) NO: 5) NO: 68) (SEQ  (SEQ  GAGTGGa ID  IDcccagga NO: NO:  (SEQ ID 67) 51) NO: 63) 22449 RSDHLST RSDNLTR DSSNLSRLRFNLSN RSAN RNDD tcTCGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID LAR RKKGAATGAC NO: 64) NO: 65) NO: 69) NO: 70) (SEQ  (SEQ  GAGTGGa ID IDcccagga NO: NO:  (SEQ ID 67) 51) NO: 63) 22454 RSDHLST RSDNLTR DSSNLSRLHFQLTG RSAN RNDD tcTCGGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID LAR  RKKGAATGAC NO: 64) NO: 65) NO: 69) NO: 71) (SEQ  (SEQ  GAGTGGa ID IDcccagga NO: NO:  (SEQ ID 67) 51) NO: 63) 25814 RSDVLSA DRSNRIK RSDVLSEQSGNLAR QSGS N/A ccGTAGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID LTR ACTGGACNO: 59) NO: 60) NO: 61) NO: 20) (SEQ  TTGacag ID  cggaagt NO: (SEQ ID62) NO: 58) 25818 DRSNLSR LKFALAN RSDVLSE QSGNLAR QSGS N/A ccGTAGA(SEQ ID (SEQ ID (SEQ ID (SEQ ID LTR  ACTGgaC NO: 5) NO: 72) NO: 61)NO: 20) (SEQ  TTGACag ID cggaagt NO: (SEQ ID 62) NO: 58) 25820 RSDVLSADRSNRIK RSDVLSE QSGNLAR QSGA N/A ccGTAGA (SEQ ID (SEQ ID (SEQ ID (SEQ IDLAR  ACTGGAC NO: 59) NO: 60) NO: 61) NO: 20) (SEQ  TTGacag ID cggaagtNO: (SEQ ID 73) NO: 58) 25822 RLSVLTI DRANLTR RSDVLSE QSGNLAR QSGA N/AccGTAGA (SEQ ID (SEQ ID (SEQ ID (SEQ ID LAR ACTGGAC NO: 74) NO: 75)NO: 61) NO: 20) (SEQ  TTGacag ID cggaagt NO:  (SEQ ID 73) NO: 58)

Example 5: ZFN Activity In Vitro

The ZFNs described in Tables 5 and 6 were used to test nuclease activityin K562 cells. To test cleavage activity, plasmids encoding the pairs ofhuman TCR-specific ZFNs described above were transfected into K562cells. K562 cells were obtained from the American Type CultureCollection and grown as recommended in RPMI medium (Invitrogen)supplemented with 10% qualified fetal bovine serum (FBS, Cyclone). Fortransfection, one million K562 cells were mixed with 2 μg of thezinc-finger nuclease plasmid and 100 μL Amaxa Solution V. Cells weretransfected in an Amaxa Nucleofector II™ using program T-16 andrecovered into 1.4 mL warm RPMI medium+10% FBS.

Genomic DNA was harvested and a portion of the TCR locus encompassingthe intended cleavage site was PCR amplified using the Accuprime HiFipolymerase from Invitrogen as follows: after an initial 3 minutedenaturation at 94° C., 30 cycles of PCR were performed with a 30 seconddenaturation step at 94° C. followed by a 30 second annealing step at58° C. followed by a 30 second extension step at 68° C. After thecompletion of 30 cycles, the reaction was incubated at 68° C. for 7minutes, then at 4° C. indefinitely.

The genomic DNA from the K562 TCR-specific ZFN-treated cells wasexamined by the Cel-I assay as described, for example, in U.S. PatentPublication Nos. 2008/0015164; 2008/0131962 and 2008/0159996.

The TCR beta locus in K562 cells has two functional copies with highsequence similarity (TRBC1 and TRBC2) which are both targeted by TCRbeta specific ZFNs. See, FIG. 4B. Thus, initially, PCR primers thatwould specifically amplify the regions around the intended ZFN cleavagesites specifically from either the TRBC1 or the TRBC2 genes were used toseparately analyze NHEJ activity following ZFN driven cleavage for bothgenes. Results are presented in Table 7 below for ZFNs pair 16787 and16783.

TABLE 7 NHEJ activity for pairs of TCR beta-specific ZFNs: analysis ofTRBC1 and TRBC2 NHEJ in TRBC2 NHEJ in TRBC1 TRBC2 ZFN1 ZFN2 % NHEJ TRBC1ZFN1 ZFN2 % NHEJ 16787 16783 8.68 16787 16783 8.73 GFP 0.00 21 GFP 0.00Mock 0.00 22 Mock 0.00 Water 0.00 23 Water 0.00 control control

The data presented in Table 7 demonstrate that the ZFNs cleave the TRBC1and TRBC2 genes essentially equally.

In addition, we tested persistence of ZFN mediated modification of TRBCin K562 cells by harvesting samples at 3 and 10 days after transfection.Results are presented in Table 8 below and demonstrate that with the ZFNpair, 16787 and 16783, target gene modification is stable 10 daysfollowing transfection.

TABLE 8 TCR beta-specific ZFNs in K562 cells ZFN 1 ZFN 2 % NHEJ 2244916783 20.1 Day 3  22454 16783 17.7 16787 16783 12.1 GFP 0.0 22409 1678314.7 Day 10 22449 16783 8.1 22454 16783 12.1 16787 16783 15.6 GFP 0.0

Several ZFN pairs targeting TRBC were analyzed for NHEJ activityfollowing varying amounts of input ZFN (either 0.4 or 0.1 μg of eachZFN). As shown in FIG. 5 , all ZFN pairs tested exhibited high activity.In this experiment, the cells were treated with a 30° C. incubationperiod following transduction with the ZFNs (see U.S. Pat. No.8,772,008). Following analysis of TCR beta-specific ZFN cleavage in K562cells, several ZFN pairs were tested in either CD4+ or CD8+ mature Tcells. Briefly, CD8+ or CD4+ cells were purchased from AllCells and werecultured in RPMI+10% FBS+1% L-Glutamine (30 mg/mL)+IL-2 (30 μg/mL,Sigma) and allowed to rest for 4-24 hours.

Lentiviral vectors were constructed containing the ZFN pairs ofinterest. They were generated from the HIV derived self-inactivatingvector construct and packaged using an HIV integrase carrying the D64Vmutation and pseudotyped with the VSV-G envelope as described above. TheAd5/F35 adenoviral vectors were generated as described previously(Perez, et al. (2008) Nature Biotechnology 26:808-816) after cloning thetwo sets of ZFNs using a 2A sequence and a cytomegalovirus internalpromoter. See, e.g., Holst J., et al. (2006) Nat Protoc. 1(1):406-17.1e6 cells/nucleofection were used with the Amaxa™ Nucleofection kit asspecified by the manufacturer for each transduction. Cells wereactivated 12-24 hours post nucleofection with anti-CD3/CD28 beadsaccording to manufacturer's protocol (Invitrogen) and grown in IMDM(GIBCO-BRL), 10% FCS media supplemented with 5 ng/mL of IL-7 and IL-15(Peprotech).

Cells were harvested 3 days after nucleofection and gene modificationefficiency was determined using a Cel-I assay, performed as described inInternational Patent Publication No. WO 07/014275. See, also,Oleykowski, et al. (1998) Nucleic Acids Res. 26:4597-4602; Qui, et al.(2004) BioTechniques 36:702-707; Yeung, et al. (2005) BioTechniques38:749-758. Several of the ZFN pairs had good activity as measured bythe Cel-I assay (NHEJ from 4-11.9%).

TCR-α-specific ZFNs were also tested in vitro as described above. Thecells were incubated at 37° C. for 1 day following the transductionprior to shifting the incubation temperature to 30° C. as describedabove. See, U.S. Patent Publication No. 2011/0129898. These ZFNs targetthe TRAC gene, results of a Cel-I assay performed on K562 cells thatreceived various combinations of these ZFNs as described above showedhigh activity. See, FIG. 6 .

Example 6: Disruption of TCR-β in Cells

The TCR-β-specific ZFNs were then used in experiments to specificallytarget the TCR locus. Initial experiments were designed to disrupt theTCR locus in Jurkat cells. TCR-β-specific ZFNS 16783 and 16787 wereintroduced on integrase-defective lentiviral vectors (IDLV) totransiently express the TRBC-targeting ZFNs. Transductions wereperformed with 0.25 μg or 0.5 μg doses of IDLV, based on measurements ofHIV Gag p24 in the vector preparations, 48 hours after activation.Vector infectivity ranged from 1 to 5×10⁴ transducing units/ng p24 byvector DNA titration on 293T cells. Cells were then assayed by FACSanalysis for loss of the CD3 marker and CD3(−) cells were enriched usingLD columns with anti-CD3 MACS Microbeads (Miltenyi Biotec) according tothe manufacturer's instructions.

As shown below in Table 9, following transduction with the ZFNs, therewas a vector dose-dependent abrogation of cell surface expression of theTCR/CD3 complex reaching up to 20% of treated cells.

TABLE 9 Loss of CD3 signal in Jurkat cells treated with TCR-β specificZFN IDLVs Untransformed 0.25 μg IDLV 0.5 μg IDLV Percent CD3(−) 2.7 13.420.2

A Cel-I assay was performed and confirmed these results with up to 26%of the TRBC alleles (18% TRBC1 and 8% of TRBC2) disrupted in the ZFNtreated cells (see FIGS. 7A and 7B, “Bulk”).

Next, The TRBC ZFNs (16783 and 16787) were introduced into primary humanT lymphocytes, and a similar level of CD3 disruption was observed byFACS, as seen in Jurkat cells. Peripheral blood T cells were harvestedfrom healthy donors and activated with CD3 and CD28 conjugated beads. 48hours post activation the cells were exposed to increasing doses ofIDLVs containing the TRBC-specific ZFNs. The cells were then cultured inthe presence of low dose (5 ng/mL) IL-7 and IL-15 to promote cellsurvival and growth. In the primary lymphocytes, up to 7% of the treatedcells were CD3 negative while almost no CD3(−) cells were observed inthe untreated control and the data is presented below in Table 10.

TABLE 10 Loss of CD3 signal in primary human T lymphocytes treated withTCR-β specific ZFN IDLVs UT 2.5 μg IDLV 5 μg IDLV 18.5 μg IDLV PercentCD3(−) 0.17 2.94 3.26 7.07

Sorted CD3(−) lymphocytes could be expanded and survived over time inthe presence of IL7 and IL15 (see FIGS. 7C and 7D), where percentmodification is indicated in FIG. 7D. FIG. 7E further demonstrates thatthe CD3(−) cells persist in the population for at least 45 days and alsoshow that the percent of CD3(−) cells in the population stays fairlyconstant over that time period. The CD3(−) cells do not appear torespond to non-specific mitogen stimulation since, PHA stimulationresults in a decrease in the percent of CD3(−) cells in the pool due toexpansion of the CD3(+) lymphocytes (FIG. 7F). This result demonstratesabsence of CD3 functional signaling in the CD3(−) cells. No phenotypicdifferences were observed in the CD3(+) and CD3(−) lymphocytes whichdisplayed a similar CD4/CD8 ratio. CD3(−) cells also maintain a centralmemory phenotype since they remain positive for CD62L, CD28 and IL-7RA(see Table 11 below).

TABLE 11 CD3(−) cells maintain a Central Memory Phenotype- percent oftotal fluorescence CD3(−) CD3(+) CD62L(+)/CD3(−) 97.6 0 CD62L(+)/CD3(+)1.25 98.4 CD62L(−)/CD3(−) 1.11 0 CD62L(−)/CD3(+) 0 1.61 CD28(+)/CD27(−)4.66 3.23 CD28(+)/CD27(+) 93.4 94.7 CD28(−)/CD27(−) 0.87 0.68CD28(−)/CD27(+) 0.97 1.43 IL-7RA(+)/CD8(−) 38.8 40.7 IL-7RA(+)/CD8(+) 4747 IL-7RA(−)/CD8(−) 3.83 2.84 IL-7RA(−)/CD8(+) 10.4 9.42

Memory T lymphocytes are less dependent upon TCR signals for homeostaticproliferation than naive T cells; we thus investigated whetherhomeostatic cytokines could promote survival and growth of previouslyactivated cells, in the absence of TCR expression. Remarkably, theTRBC-ZFNs treated cells could be expanded in culture by supplementationwith low dose IL-7 and IL-15, with the proportion of CD3(−) cellsremaining stable for more than 50 days in the absence of TCR triggering.Thus, ZFN exposure was well-tolerated in primary lymphocytes andresulted in the stable disruption of the targeted TRBC gene. Therefore,CD3(−) cells were sorted to near purity and further expanded with IL-7and IL-15 for more than 3 weeks with growth rates similar to CD3(+)cells, demonstrating that homeostatic cytokines do not require TCRsignaling functions to promote survival/proliferation of previouslyactivated cells.

These data demonstrate the successful generation of a novel populationof CD8 T cells with phenotypic characteristics of TCM but with surfaceexpression of the endogenous TCR permanently disrupted.

Example 7: Introduction of aWT-1 Specific TCR in Cells that hadPreviously had the Endogenous TCR Permanently Disrupted

CD3(−) T lymphocytes were sorted after treatment with the TCR β-specificZFNs and a lentivirus used to randomly integrate the WT1-TCR β transgeneas described in FIG. 1 (49.5±30% mean±SD transduction efficiency, n=4).Thus, in TCR-β-edited cells, expression of the transferred WT1-TCR froman integrated vector rescued surface translocation of CD3 (FIG. 8 ,1^(st) row).

In contrast to unedited TCR-transferred lymphocytes in which there wasno inherent growth advantage to expression of the introduced TCR (FIG. 8, 2^(nd) row) with respect to the untransduced cells on polyclonalexpansion, TCR β chain disrupted cells containing the WT1-TCR could beenriched to >90% purity by polyclonal stimulation, indicating thatsurface expression of the transferred TCR/CD3 complex in TCR-β-editedcells was necessary and sufficient to promote TCR-mediated expansion ofgenetically modified cells (FIG. 8 , 1^(st) row). The exogenous WT1-TCRVβ chain (Vβ21) was expressed in TCR-β chain disrupted lymphocytes atapproximately 2-fold higher mean levels than in unedited TCR-transferredcells and reached expression levels similar to those of the endogenousVβ21 chain of control T cells and was stably maintained in culture (FIG.9A and FIG. 9B). Accordingly, after transduction with the same dose ofPGK-WT1 LV, up to 22% of TCR-β-edited lymphocytes bound the WT1₁₂₆₋₁₃₄pentamer as compared to only 2.6% of unedited cells. (FIG. 9A, lowerhistogram).

Thus, in the absence of competition from the endogenous TCR β chain,surface expression of the transgenic TCR β chain reaches physiologicallevels. To verify the function and avidity of TCR-β-edited lymphocytes,we compared TCR β chain disrupted cells with unedited cells transducedwith the same PGK-WT1 LV for the ability to lyse HLA-A2⁺ targets pulsedwith increasing WT1₁₂₆₋₁₃₄ peptide concentrations (see FIG. 9C). Thisfunctional assay measures activity by a ⁵¹Chromium release assay forlysis of labeled T2 cells pulsed with increasing concentrations of theWT1₁₂₆₋₁₃₄ HLA-A2 restricted peptide, or with the irrelevant CMV-derivedpp65₄₉₄₋₅₀₃ HLA-A2 restricted peptide (10 μM, Proimmune) as a negativecontrol, at an Effector/Target (E/T) ratio of 12.

Edited T cells were stimulated and 3 weeks later were tested forrecognition of the labeled T2 cells by co-incubation for 5 hours. TCR βchain disrupted cells (denoted TCR-edited in FIG. 9C) killed targetsmore effectively than unedited (denoted TCR-transferred) WT1 LVtransduced cells (EC50: edited cells: 90.51 nM, with 95% CI:48.84-167.7; unedited TCR-transferred cells: 269.1 nM, with 95% CI:175.1-413.5), likely reflecting the higher frequency and expressionlevel of the transgenic WT1 TCR in the TCR-β edited samples. EC50 wascalculated by non-linear regression analysis of ⁵¹Chromium release databy using the sigmoidal dose-response equation of the GraphPad PrismSoftware.

Results are represented as average SD of % lysis (*=p<0.05, **=p<0.01,TCR-edited n=6, TCR transferred, n=4). To assess reactivity at a singlecell level, cell were analyzed for Vβ21 expression (see Table 12 below)which showed that, despite fairly equal copy number of the vectors, Vβ21expression was greater in the TCR-edited cells.

TABLE 12 TCR expression and vector copy number/cell in TCR transferredand TCR edited lymphocytes Disruption endogenous PGK-WT1 LV β chain byor Vβ21 % Vβ21+ TRBC-ZFN EF1α-WT1 LV RFI* cells CpC^(§) TCR- No EF1α-WT1LV 0.41 36.7 1.9 transferred PGK-WT1 LV 0.54 62.7 2.1 TCR-edited YesPGK-WT1 LV 0.91 97.3 1.2 UT No None 1 3.2 0 *TCR expression was measuredby flow cytometry and was plotted as relative fluorescence intensity(RFI = Vβ21 MFI of transduced cells/Vβ MFI of untransduced cells).^(§)Vector copy per cell (CpC) was measured by quantitative PCR asdescribed (Kessels et al. (2001) Nature Immunol 2(10): 957-61).

To assess alloreactivity at a single cell level, clones were isolatedand expanded from both TCR-β edited and TCR-transferred cells,previously sorted for WT1₁₂₆₋₁₃₄ pentamer binding to enrich for cellsdisplaying optimal exogenous TCR expression. Clones were exposed to T2cells pulsed with 10 nM of the WT1₁₂₅₋₁₃₄ HLA-A2 restricted peptide(left panel) or to allogenic PRMC (right panel) at astimulator/responder ratio of 1. The number of specific spots is shownon the y axis as the number of spots produced in the presence ofstimulators minus the number of spots produced by effectors alone(**=p<0.01). TCR β-edited clones displayed reduced alloreactivity,compared to TCR-transferred cells (see FIG. 10 , compare the 10A to10B), possibly reflecting the reduced risk of TCR mispairing in theabsence of one endogenous TCR chain.

These data demonstrate the functional advantage offered by expression ofa tumor specific exogenous TCR in a host CTL with abrogated endogenousTCR-β chain expression.

Theoretically, surface re-expression of the unedited endogenous TCR αchain may still occur in TCR-β edited cells, following TCR genetransfer. To directly assess the potential for misparing in TCR-β chaindisrupted lymphocytes, CD3(−) cells were transduced with a LV encodingonly the WT1-specific TCR β chain gene and the ΔLNGFR marker(WT1-β-ΔLNGFR-LV). Transduction efficiency was assessed as a percentageof the ΔLNGFR^(pos) lymphocytes (see FIG. 11 ). Vβ21 expression wasmeasured on ΔLNGFR^(pos) cells. The mean fluorescent intensity (MFI) ofVβ21 is indicated. Despite the absence of WT1-specific α chain, Vβ21expression was detected in up to 83% of ΔLNGFR^(pos) TRBC-disruptedcells, demonstrating that even a cysteine-modified TCR β chain insertedinto a cell with a TRBC disruption is capable of mispairing with theendogenous TCR α chain.

Next, CD3(−) lymphocytes are used to introduce the WT1-TCR β donorconstruct into the endogenous TCR locus. The donor is constructed asdescribed above and used in conjunction with the TCR-β-specific ZFNs tocause integration of the TCR-β transgene at the endogenous locus. Thecells become positive for both CD3 and the VB 21.3 TCR β chain.

Example 8: Disruption and Targeted Integration of the TCR-α Chain

To eliminate the potential for TCR chain mispairing, we designed a pairof ZFNs targeting the constant region of the TCR α chain (TRAC) gene(FIG. 6 ) and obtained TCR-α-edited T lymphocytes (see FIG. 12A),following the same protocol described to TCR-β editing and obtainedTCR-α-edited T lymphocytes, following the same protocol described forTCR-β-editing (FIG. 12B, 12C, 13 ). To design a complete a/P TCR editingprotocol that permits rapid isolation of engineered cells at each stepof chain disruption/replacement, we generated a set of LV carrying asingle α or β WT1-specific TCR chain, and used IDLV or adenoviralvectors (AdV) to transiently express TRBC- or TRAC-targeting ZFNs inlymphocytes (FIG. 14 for timeline and representative flowconditions/results for full TCR editing)

CD3(−) cells were efficiently generated with every ZFN-containing vectortested and sequencing at the site of nuclease cleavage reveals the smallinsertions and deletions (indels) present after repair by NHEJ (FIG. 13). AdVs, which proved more efficient in mediating TCR gene disruptionthan IDLVs, were selected for the purpose of complete TCR editing. Tcells harvested from healthy donors were first exposed toTRAC-ZFN-Ad5/F35 48 hrs post-activation with baCD3/CD28, cultured in thepresence of IL-7 and IL-15, and the resulting CD3(−) cells isolated bysorting were transduced (49±29& mean±SD transduction frequency, n=3)with a LV encoding the WT1-α chain (WT1-α LV).

Cells with rescued CD3 expression were then sorted, stimulated withbaCD3/CD28 for one cycle, and then exposed to TRBC-ZFN-Ad5/F35. Thesecond round of ZFN exposure yielded up to 23±4% newly CD3(−) cells,indicating that primary T lymphocytes are permissive to multiple roundsof ZFN manipulation. The CD3(−) cells were sorted and transduced (18±7%mean±SD transduction efficiency, n=3) with a WT1 TCR-β chain LV.Expression of the transferred WT1-β chain again rescued surfacetranslocation of CD3, which was now co-expressed in balanced proportionwith the WT1-TCR Vβ chain in TCR-edited cells (FIG. 14 and FIG. 15 ). Incontrast to unedited TCR-transferred lymphocytes, TCR-α/β disruptedcells could be enriched to near purity by polyclonal stimulationfollowing TCR gene transfer, and homogenously expressed the high levelsof WT1-specific TCR required to bind the WT1₁₂₆₋₁₃₄ pentamer (see FIG.15 ).

These results indicate that surface expression of the transferredTCR/CD3 complex in TCR-edited cells was necessary and sufficient topromote expansion of the cells with the desired specificity for WT1(FIG. 14 , right plot). Disruption of the α and β TCR chains wasconfirmed in TCR-α/β edited cells by Cel-I analysis. No phenotypicdifferences were observed in TCR-transferred and TCR α/β-editedlymphocytes, which displayed a TCM surface phenotype, as evidenced byhigh expression of CD62L, CD27, CD28 and IL-7rα. To verify the functionand allogenic response of the fully edited lymphocytes, TCR α/β-editedand TCR transferred lymphocytes were polyclonally stimulated.

Three weeks after polyclonal stimulation, TCR-α/β-edited and TCRtransferred lymphocytes were exposed to either i) T2 cells pulsed withincreasing concentrations of the WT1₁₂₆₋₁₃₄ HLA-A2 restricted peptide,or with the irrelevant CMV-derived pp65₄₉₅₋₅₀₃ HLA-A2 peptide (see FIG.16A) or ii) WT1⁺ HLA-A2⁺ (black in FIG. 16B) or HLA-A2⁻ (grey) leukemiccells harvested from AML patients with (dashed symbols) or without (fullsymbols) pulsing with WT1₁₂₆₋₁₃₄ peptide (50 nM). FIG. 16C shows similarresults where allogenic PBMC were used as target. All assays wereperformed at a stimulator/responder ratio of 1. Specific spots are shownon the y axis as spots produced in presence of stimulators minus spotsproduced by effectors alone. *=p<0.05, **=p<0.01, ***=p<0.001.

Example 9: Potential Off Target Cleavage Analysis

In silico analysis was used to identify the most likely potentialoff-target cleavage sites for both the TRAC- and TRBC-specific ZFN pairsas described in Perez, et al. (ibid). Sites were identified thatcontained up to 10 recognition site mismatches for either heterodimerZFN pairs or homodimer pairs, although the most likely potential offtarget sites for these ZFN pairs were all targets for ZFN homodimers.The most likely potential off target sites identified are shown below inTables 13 (TRAC) and 14 (TRBC).

TABLE 13 Potential off target sites for TRAC- specific ZFNs Chro-   La-mo- Start # mis- bel some site Sequence matches Gene OT1 20  20683361AGGCACAaGC  6 RALGAP AAtGTCACAA A2 GtACcaTGCt TGTACTT (SEQ ID  NO: 76)OT2  6  10525974 AGGTACAaGt  5 GCNT2 AAAGaCGTAT GaACTTTGCt TGTACTT (SEQ ID NO: 77) OT3  X 135000000 AAaTACAaGC  6 — cAAGcCAAGG TGgCTTTGCGTGTAaAT (SEQ ID  NO: 78) OT4 18  60239118 ATaTACAatt  8 ZCCHC2AAAGTCAGCT TTtACTTTGC  agtTACTT (SEQ ID  NO: 79) OT5  7  48500931TAGaACAtcC  6 ABCA13 AAAcTCTGGA CCGACTTTGC aTGTcCAG (SEQ ID  NO: 80) OT6 7 141000000 ATtCAaACaC  7 — AAAGTCCCGT GGAtTTTGCt TtTAaAT  (SEQ IDNO: 81) OT7  8   2463159 ATGCAggaGC 10 — AAgGTCACTC TGACcTTcCt  TtgcCTT(SEQ ID  NO: 82) OT8 18   4312947 ATGCACACaC  7 — AAAcTCATTT AagCTTTGCt TtTcCAT (SEQ ID  NO: 83) OT9 11  70854569 CAGCcCAtGg  7 SHANK2AAtGTCATTC TcACaTTGCt  TGTGCTT (SEQ ID  NO: 84) OT1 13  57970961AAGCAaAaGa  8 — 0 AAAaTCAATA TGACTTgGCt  TtgGCTT (SEQ ID  NO: 85) OT1  2 69188623 AAGgtCACtC  7 — 1 ActGTCTGTG TGGAgTTTGC GTGTcCTC (SEQ ID NO: 86) OT1  X  78538296 AAGCAggaGC  8 — 2 AAAGTCACAT CTtACaTTGCGgcgGCAT (SEQ ID  NO: 87) OT1  2 108000000 ATGTAattcC  8 — 3 AAAGTCCTCCATGACcTgGC  tTcTACCT (SEQ ID  NO: 88) OT1  8  28249779 CTaCAaAttC  8 — 4AAtGaCAGTA GAGACTTTGC  tTtTACTT (SEQ ID  NO: 89) OT1  9  93810846ATGCAacaGC 10 — 5 AAgagCAGCA TGACTTTGtt  TtTcCTT (SEQ ID  NO: 90) TRA 14 23016627 GTGCTGTgGC  4 TRAC C CTGGaGcAAC AAATCTGACT TTGCaTGTGC AA (SEQ ID  NO: 91)

TABLE 14 Potential off target sites for TRBC- specific ZFNs chro- La-mo- Start  # mis- bel some site Sequence matches Gene C1  1 236659757CCcAagCCAG 6 — ggCTACTGCT GGGTgGAACT GGACATGC (SEQ ID  NO: 92) C10 10 90573967 CCcTGTgCgG 7 LIPM TTCTgCTTAA CAGTAGAACa GGACActT (SEQ ID NO: 93) C5  5 165037707 ACATGTCaga 5 — TTCTACATGA GGTAGAACTG  ttCTTGT(SEQ ID  NO: 94) C2  2  71186796 ACAAGggCAG 7 ATP6V1B cTCTgtCCAA 1GGTAGcACTG  GgCCTGT (SEQ ID  NO: 95) C15 15  75401377 CgATGTCCAG 6 —aTgTACCTCA GGaAGgACTG GcCCTGG (SEQ ID  NO: 96) C3  3 159730398CCAAGTCCtc 6 — cTCTAgGAAG GGGTAGAACT GGAaTTtG (SEQ ID  NO: 97) C1.2  1 60766812 GaAGGTCCAG 7 — TgCaAtGTTG AaTAGAAgTG  GACATcT (SEQ ID  NO: 98)C17 17  11136639 AgAGGcCCAc 7 — TcCTAgAAGG GGTAGAcCTG GAtCTGG (SEQ ID NO: 99) C15. 15  67440002 CCAGGTCCAG 6 SMAD3 2 TTCTACCAGC CacAGAtgTG agCATGT (SEQ ID  NO: 100) C2.2  2 120313989 ACAAtTCCAG 7 PCDP1TTCaAgAATC TTtTAaAggT  GGACATGG (SEQ ID  NO: 101) C6  6 166419926GCtGGTgCAG 7 — cTCTACACGG ATGcAGAgCT GGtCCTcC (SEQ ID  NO: 102) C2.3  2114733178 CCtGGgCCAG 8 — TgCTgCTTGT CcTtGAACcG  GgCCTGG (SEQ ID NO: 103) C7.2  7   4224762 GgAGaTCCAG 8 SDK1 TgCgACAGTC AGaAGAAggGGACTcGG (SEQ ID  NO: 104) CX  X  73070675 CTaCAaAttC 8 XIST AAtGaCAGTAGAGACTTTGC  tTtTACTT (SEQ ID  NO: 105) CX.2  X 130414175 CCAGGTCagG 8AGSF1 TTCcggAAAG AAGTAGAACT  tGACCccT (SEQ ID  NO: 106) TRB  7 142499011TCAAGTCCAG 2 TRBC C TTCTACGGGC TCtCGGAGaA TGACGAGTGG  A (SEQ ID NO: 107)

As can be seen from the data shown in FIGS. 17 and 18 , there are noadditional bands present in the off-site samples that have been treatedwith the ZFNs as compared to those that have not been transduced withthe ZFN expression vectors (also compare with the TRAC and TRBC loci).Note that for technical issued relating to the PCR of the target, theC2.2 potential off target has not yet been fully analyzed. Nevertheless,it appears that the TRAC- and TRBC-specific ZFNs have a great deal ofspecificity for their intended targets.

1-11. (canceled)
 12. A method of inactivating a TCR gene in a cell, the method comprising integrating a transgene into the TCR gene by a zinc finger nuclease comprising a zinc finger protein that binds to a target site in the TCR gene, wherein the target site is within any of SEQ ID NO: 1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 or 63, and further wherein the binding of the zinc finger protein at the target site in the TCR gene results in cleavage of the TCR gene such that the TCR gene is inactivated by integration of the transgene.
 13. The method of claim 12, wherein the transgene encodes a chimeric antigen receptor (CAR).
 14. The method of claim 12, wherein the TCR gene is an endogenous TCRα or TCRβ gene.
 15. The method of claim 14, wherein the transgene is integrated into exon 1 or exon 3 of the endogenous TCRα gene.
 16. The method of claim 12, wherein the cell is a T-Cell.
 17. The method of claim 12, wherein the cell is a stem cell.
 18. The method of claim 17, wherein the cell is a hematopoietic stem cell.
 19. A method of treating cancer, infections, autoimmune disorders or graft-versus-host disease (GVHD) in a subject in need thereof, the method comprising introducing into the subject an isolated cell or a cell line comprising an inactivated endogenous T-cell receptor (TCR) gene, wherein the TCR gene is inactivated by integration of a transgene into the TCR gene by a zinc finger nuclease comprising a zinc finger protein that binds to a target site in the TCR gene, wherein the target site is within any of SEQ ID NO: 1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 or 63, and further wherein the binding of the zinc finger protein at the target site in the TCR gene results in cleavage of the TCR gene such that the TCR gene is inactivated by integration of the transgene.
 20. The method of claim 19, wherein the transgene encodes a chimeric antigen receptor (CAR).
 21. The method of claim 19, wherein the TCR gene is an endogenous TCRα or TCRβ gene.
 22. The method of claim 21, wherein the transgene is integrated into exon 1 or exon 3 of the endogenous TCRα gene.
 23. The method of claim 19, wherein the cell is a T-Cell.
 24. The method of claim 19, wherein the cell is a stem cell.
 25. The method of claim 24, wherein the cell is a hematopoietic stem cell. 